The introduction of sample into a high pressure liquid chromatography (“HPLC”) analysis system is normally accomplished through the operation of an apparatus termed a sample injector, which is interposed in the HPLC system fluid circuit downstream of the mobile phase delivery pump(s) and upstream of the analytical column. This configuration accomplishes a substantially direct introduction of sample to the head or inlet end of the analytical column. Variants of this simple configuration are known in the art, an example being a “sample trapping” configuration where the sample injector may reside between an auxiliary pump and a dedicated sample trapping column, and where the sample trapping fluid circuit may be isolated from the analytical column fluid circuit during the trap loading interval. At the completion of the trap loading process, the trap column is then switched into the analytical column fluid circuit by the action of one or more switching valves. Following the switching step, sample is eluted from the trapping column onto the analytical column by the analytical mobile phase, thereby enabling analyte separation to be performed on the analytical column substantially as in the direct introduction mode. The sample trapping mobile phase may be selected to differ in composition and flow rate from the analytical mobile phase, as is known in the art. Similarly, the stationary phase used in the sample trapping column may be selected to differ from that used in the analytical column, also as known in the art.
In any case, the normal operation of the sample injector is to cause a liquid volume, which initially resides at a pressure substantially equal to atmospheric pressure, to be introduced into a mobile phase stream which is flowing, and which is at a relatively high pressure, typically in the range of thousands of pounds force per square inch (PSI) (tens of megaPascals (MPa)). The liquid volume which is introduced is typically a prescribed volume of a sample solution in which one or more sample analytes are dissolved. In some implementations, the sample volume may be bracketed by small air-gaps, which are utilized to minimize the broadening of the sample zone as the sample is conveyed within a fluid conduit from a sample vial toward or to the point of injection.
The actual injection operation which introduces a liquid sample volume into a high pressure mobile phase delivery stream is typically carried out by the action of one or more valves capable of redirecting the mobile phase flow. Two general approaches to sample injector implementation have been widely utilized over the past several decades. One approach is the multi-port rotary shear seal valve, commercially available from several vendors including Valco Instruments Company Inc. (VICI, Houston, Tex.), Upchurch Scientific Division of Scivex Corporation (Oak Harbor, Wash.), and Rheodyne LLC (Rohnert Park, Calif.). The other general approach is the resistive bypass injector configuration, in both manually-actuated and automated forms.
A six-port rotary shear seal valve provided with an external sample loop is a common configuration used to perform sample injection in conventional HPLC. The use of such a valve is promoted by its relatively small overall size, moderate cost, and relative simplicity of operation. In common with many other multi-port shear seal valves, the conventional six-port rotary shear seal injection valve integrates multiple stream-switching elements into a single rotor-stator assembly, thereby enabling coordinated actuation of the plurality of valve elements by a single actuating mechanism such as an electric motor.
Related to the six-port, external-loop shear seal valve is another common variant, the four-port, internal-loop valve, which shares with the six-port valve the desirable attributes of small size, moderate cost, and simplicity of operation. The reduction in external port count from six to four is a result of the sample loop being implemented internal to the valve, thereby eliminating the need for the two external ports used for the attachment of an external sample loop.
Both the four-port and six-port valve types, when used as sample injection valves, are essentially two-state devices. In the LOAD state, fluid communication between the mobile phase pump and the column is enabled while the sample loop is maintained in fluid communication with a sample aspiration path. In the INJECT state, fluid communication between pump and column is established through the sample loop, enabling the transport of the loop contents toward the column. An undesirable attribute shared by conventional rotary shear seal injection valves is that during transition between the LOAD and INJECT state, or between the INJECT and LOAD state, the flow path between pump and column is temporarily blocked. This behavior is often described as a “break-before-make” type of switching.
Even at conventional HPLC flow rates and pressures, break-before-make switching of the fluid path internal to the sample injection valve gives rise to very significant flow and pressure transients in the mobile phase stream. These transients arising from flow blockage and reinstatement occur during both the INJECT-to-LOAD and the LOAD-to-INJECT valve state transitions. During the time interval in which the pump-to-column flow is obstructed, the pump continues to source mobile phase into a now dead-ended fluid path, causing the liquid pressure in the fluid circuit upstream of the obstruction to rise. At the same time, the column is continuing to sink mobile phase from the fluid circuit downstream of the blockage, in accordance with the pressure differential imposed across the column, and the resistance to flow exhibited by the column. In circumstances where a meaningful additional resistance to flow exists between the column outlet and the ultimate liquid discharge point (typically discharging to atmosphere downstream of a detector), the sink flow rate will be determined by the sum of the column and additional series resistances, and the pressure differential asserted over that collective resistance. For a conventional rotary shear seal injection valve with a typical motorized actuator, the timeframe during which the upstream and downstream fluid circuits are isolated may readily be in the range of approximately one hundred to several hundred milliseconds.
Because of the relative stiffness of liquids, a flow blockage of approximately one hundred to several hundred milliseconds duration can lead to a significant pressure differential being imposed across the injection valve. Specifically, the upstream and downstream fluid circuits may depart from one another in internal pressure at rates corresponding to tens of thousands of PSI per second (hundreds of MPa per second). When the valve nears completion of its state transition, the previously-isolated upstream and downstream fluid circuits are restored to a condition of fluid communication. The reinstatement of fluid communication within the valve is immediately followed by an aggressive exchange of liquid from the higher-pressure to the lower-pressure circuit. The rate-of-change of pressure observed near the column head, during the initial convergence of pressures, may exceed one million PSI per second (7000 MPa per second), for an interval of one to several milliseconds. The initial pressure transient may be followed by one or more cycles of damped ringing attributable to the combined effects of fluid inertance, fluid capacitance, and resistance to fluid flow.
These transients typically become more severe as system operating pressure increases, or as the pre-column system volume decreases (the latter tending to reduce the parasitic fluid capacitance available to cushion the effect). Ongoing interest within the HPLC community in achieving higher separation efficiencies and shorter analysis times has led to the development and commercial adoption of column packing particles as small as approximately 1.5 micrometers in mean diameter. Effective use of analytical columns packed with such small particles typically requires system operation in a pressure regime above that of conventional HPLC, commonly termed very high pressure liquid chromatography (“VHPLC”). Conditions associated with VHPLC system operation, which may include mobile phase delivery pressures of 10000 PSIG (70 MPa) or higher, and very low pre-column system volume, are substantially the conditions which exacerbate the transient.
Abrupt transients of liquid flow rate and liquid pressure in the vicinity of the column head can be extremely disruptive to HPLC columns. The disruptive effects upon the column are most readily observed as a loss of achievable separation efficiency for a defined separation which is performed repeatedly, or as a change (typically an increase) in the resistance to flow exhibited by the column, for a defined operating condition corresponding to a prescribed mobile phase composition, flow rate, and column temperature.
Modifications to known rotary shear seal injection valves include a feature intended to reduce the time duration of the flow blockage during valve state transition. In these valves a groove or channel extends from the pump port on the valve stator toward the column port, whereby the connection time between the pump and the column is prolonged or extended over the connection time associated with an unmodified valve undergoing state transition. In one commercial embodiment, a typical time duration of flow blockage of about 50 milliseconds is achieved. Unfortunately, in VHPLC system operation, even a 50 millisecond flow blockage can lead to dramatic column impairment. The transients arising from intermittent flow blockage are typically rapid events with characteristic timeframes measured in the range of one to several milliseconds, and can be produced by flow blockages of similarly short duration. In that context, a 50 millisecond break-before-make switching behavior is highly significant, and a valve exhibiting that duration of complete flow blockage is substantially inadequate to address the column degradation problem.
The resistive bypass sample injection technique of Abrahams and Hutchins circumvents the direct blockage of flow during sample injection by providing a parallel fluid pathway between pump and column which is never obstructed by valve actuation. In the LOAD state of a resistive bypass injector, pump-to-column flow is conveyed entirely through the bypass path. In this state, the sample loop is isolated by diaphragm or other type valve elements from system pressure, allowing the sample loop to be loaded with a sample residing at substantially atmospheric pressure. During transition to the INJECT state, the valves previously isolating the sample loop are opened, exposing the sample loop contents to system pressure. In the INJECT state, the fluid path encompassing the sample loop resides in parallel with the constantly-enabled bypass path. A critical aspect of the valve implementation is the selection of the relative resistance to flow of these two parallel fluid paths. In one implementation, the tubing internal diameters and lengths are selected such that the bypass path has a resistance to flow which is about 32 times higher than that of the sample loop path. With this configuration, when the valve resides in the INJECT state, approximately 97 percent of the pump flow is sustained through the sample loop, with the remaining 3 percent bypassing the sample loop. The use of less than 100 percent of the pump flow to convey the sample from the loop will cause the injected sample to emerge from the loop over a longer interval of time, and to become diluted by the bypass flow. In the example above, the sample band would emerge from the injector in a volume approximately 1.03 times larger than that which would be obtained in the absence of any bypass flow. Because the characteristic peak width of an injection impulse is an important figure-of-merit of an HPLC sample injector, effort is expended to keep the bypass percentage as low as possible in light of other design considerations. Among those considerations are the relative rates at which the sample and bypass paths are purged with fresh mobile phase. In a gradient mode of chromatography, the mobile phase compositional gradient will typically pass through the sample injector, and therefore will negotiate both the sample and the bypass paths when the valve is in the INJECT state. Poor matching of the purging rates of these two parallel paths can lead to corruption of the gradient profile.
Another consideration with the resistive bypass injection technique is that the resistance of the bypass path is additive in series to the column and other system resistances when the valve is in the LOAD state (that is, when 100 percent of the pump flow is being directed through the bypass path). When the valve is transitioned to the INJECT state, the resistance of the bypass path is largely circumvented, as that path is placed in parallel with a sample loop path having a resistance typically some thirty times lower. Thus the overall system resistance to flow can be observed to shift between two values, corresponding to the LOAD and the INJECT states of the sample injector, respectively. These shifts occur over timeframes governed by the overall system hydraulic time constant.
In addition to the above considerations, the resistive bypass injector as implemented by Abrahams and Hutchins is generally more complex and more expensive to manufacture than a conventional multi-port rotary shear seal valve.
The transients discussed above are transients arising from the temporary blockage of flow within an HPLC or VHPLC system as a result of injector actuation. An additional aspect of sample injection is the transient behavior associated with the pressurization of the sample loop contents, corresponding to the charging of the sample loop fluid capacitance. The magnitude of this transient is a function of sample loop volume, air gap volume (if used), system operating pressure, and the compositions of the sample diluent and mobile phase. Its time-course is affected by the configuration of the fluid conduits upstream and downstream of the sample injector. Whereas the transient in mobile phase flow or pressure associated with flow blockage is substantially similar for both state transitions of the sample injector (LOAD to INJECT and INJECT to LOAD), the loop charging transient is manifested in the LOAD to INJECT transition. The behavior arises from the fluid current inrush which pressurizes the sample loop contents from atmospheric pressure to system operating pressure, typically in a timeframe of one to several milliseconds.
Depending upon the configuration of the upstream and downstream fluid conduits, that inrush current may be transiently sourced from both the column head and pump capacitances. When the sample loop is taken off-line during the INJECT to LOAD transition, a rapid decompression of the pressurized loop contents occurs, but that discharge current is directed toward other system components and is substantially invisible to the column. While the resistive bypass injector configuration substantially circumvents the problem of flow blockage transients, it shares with the shear seal injection valve the undesirable characteristic of causing a loop charging transient when the un-pressurized sample is exposed to system pressure. In a VHPLC system, the loop charging transient may be the dominant transient associated with sample injection, and under worst-case conditions of a large sample loop with air-gaps present, may result in a nearly instantaneous and substantially complete depressurization of the column head, with attendant destructive impact upon the column.